Method for growing circoviruses

ABSTRACT

Methods for growing circoviruses, in particular porcine circoviruses, which are obtained from an infected cell culture after one or more passages in cultures of porcine, bovine or human cells are described. When the porcine circoviruses grow, a cytopathogenic effect occurs in the cell culture.

The invention relates to methods for growing and quantifying theinfectious or antigenic amount and determining antibodies againstcircoviruses, in particular porcine circoviruses (PCV). The inventionalso relates to methods for detecting the reduction in circoviruses andcircovirus-like viruses of humans through the method for manufacturingpharmaceuticals from biological material (1).

Porcine circoviruses are isometric non-enveloped viruses about 17 nm insize and having single-stranded circular DNA of 1.76 kb. Antibodiesagainst porcine circovirus have been found in serum from humans, miceand cattle by means of an indirect immunofluorescence detection (IFA)and by means of the ELISA method (2). Antibodies are detected in 53 to92% of slaughter pigs (3). The clinical significance of PCV is stillunknown. A new type or porcine circovirus, which leads to lesions in pigtissues and can be correlated with disease symptoms, has recently beendetected. This so-called type 2 (PCV2) differs from the aforementionedtype 1 (PCV1) mainly in terms of the pathogenicity (3 and 4).

Although porcine circovirus can be detected as contaminating agent inpig tissue cultures, it has not to date been possible to grow the virusin vitro and establish a routine test for growth and for quantitativedetection of the virus, e.g. on the basis of a cytopathogenic effect(CPE), in the virus-replicating cells (2).

However, PCV is detected in vivo by means of the polymerase chainreaction in lymph nodes and cells of the spleen, tonsils, liver, heart,lungs, nasal mucosa, kidneys, pancreas and intestine (3). Since pigorgans are used for human transplantation (5), porcine circovirus shouldbe regarded as a potential risk virus for humans.

The object of the present invention was therefore to develop a methodfor cultivating porcine circovirus in vitro in order to be able toexamine its infectivity. It was additionally intended to develop amethod for neutralizing porcine circovirus by specific antibodies andremoving it from biological materials from pigs, humans or othervertebrates, so that these materials can be employed withoutreservations directly or indirectly for therapeutic purposes, e.g. forobtaining insulin, heparin, blood and plasma, cell culture media andconstituents thereof, including trypsin, and for cells for producingrecombinant proteins. It was finally intended that successful growing ofPCV in cell cultures would also make it possible to produce a vaccine bymethods known per se. This may involve using inactivated PCT or anavirulent PCV strain (e.g. through selection of an avirulent PCV strainafter adaptation to various cell cultures and/or after treatment ofinfected cell cultures with mutagens or after genetic modification ofthe PCV) as live vaccine. In addition, the antigenic material obtainedfrom grown porcine circoviruses can also be employed for diagnosticpurposes.

This object is achieved by a method for growing circoviruses, inparticular porcine circoviruses (PCV), in which circoviruses obtainedfrom an infected cell culture grow in cultures of porcine, bovine orhuman cells after one or more passages in the cell cultures.

For the method of the invention, porcine circoviruses obtained from aPK15 (=porcine kidney) cell culture inapparently infected with PCV were,after passage in other cell cultures, in particular porcine cellcultures, grown in these cell cultures, with a cytopathogenic effectbeing displayed. The presence of porcine circoviruses was in this caseverified with the aid of a specific nested polymerase chain reaction(PCR). Primers with the following DNA sequences were employed for this:

1. PCR plus: GAG AGG AAG GTT TGG AAG AGG (946-966) (SEQ ID NO. 1)

1. PCR minus: CCA CTG GCT CTT CCC ACA ACC (1358-1338) (SEQ ID NO. 2).

2. PCR plus: GGT GAA GTG GTA TTT TGG TGC C (1025-1046) (SEQ ID NO. 3)

2. PCR minus: CTA TGA CGT GTA CAG CTG TCT TCC (1326-1303) (SEQ ID NO. 4)

These primers were selected from the origin of replication (7).

When carrying out the method of the invention, it was observed that theporcine circoviruses cannot be grown equally well in all cultures ofvarious mammalian and human cells. Growth was successful in cultures ofcells from various porcine organs, from bovine kidney, bovine lung andhuman lung. A cell culture which is very suitable for successful growthof porcine circovirus and which was developed from fetal porcine testiswas deposited on Jul. 10, 2000, under the provisions of the BudapestTreaty at Deutsche Sammiung Von Mikroorganismen und Zelikulturen GmbH(DSMZ), Mascheroder Weg 1b, D—38124 Braunschweig, German, and wasassigned Accession No. FSHO-DSM ACC 2466.

The ELISA method is very suitable for quantitative detection ofantibodies present in serum against porcine circovirus. This entailscircoviruses being incubated, after adsorption onto a support material,with the serum to be investigated and thus being bound to a primaryantibody present in the serum. A secondary labeled antibody directedagainst the primary antibody is then brought into contact therewith and,after the unbound secondary antibody has been washed out, the lightsignal emitted by the bound labeled antibody (extinction) is measured.The sandwich method known to the skilled worker is suitable fordetecting the circovirus antigen, in which case an antibody againstcircoviruses which is bound to a support material binds the antigen inthe serum to be investigated; a (labeled) antibody directed againstcircovirus antigen is then brought into contact therewith and, after theunbound antibody has been washed out, the light signal emitted by thebound labeled antibody (extinction) is measured.

Sera containing neutralizing antibodies against porcine circovirus aresuitable for neutralizing circoviruses in biological material.Neutralizing antibodies have been found in porcine sera and humanimmunoglobulin (γ-globulin). A human immunoglobulin particularlysuitable for neutralizing circoviruses in biological material is oneobtained from high-titer human donors who have a titer of specificantibodies which is at least two to three times higher than normaldonors, the average PCV antibody titer being determined on a plasma poolfrom at least 1000 donors.

The virus safety of pharmaceuticals of biological origin (e.g. fromhuman blood/plasma, from cell cultures, from animal organs/tissues) isinvestigated as required by the authorities (e.g. CPMP/BWP/268/95: Notefor guidance on virus validation studies: the design, contribution andinterpretation of studies validating the inactivation and removal ofviruses; CPMP/BWP/269/95 rev. 3: Note for guidance on plasma-derivedmedicinal products); in these so-called virus validation studies,viruses are deliberately added to material at various production stepsin the method of manufacture of biologicals, and the removal and/orinactivation of the viruses by the step in the method is determined. Theviruses used for this investigation either should possibly occur in thebiological starting material or, if these viruses cannot be grown invitro, are model viruses with physico-chemical properties as similar aspossible to the contaminating viruses. An example of a model virus forthe human circovirus TTV is PCV. In investigations of a step in themethod of manufacture of therapeutic compositions from biologicalmaterial—heat treatment at 60° C. in stabilized aqueous solution—itemerged that the porcine circovirus is labile and could be inactivatedwithin a few hours; it is thus possible to demonstrate the capacity ofthe method of manufacturing biologicals to inactivate TTV by a heattreatment. It is possible analogously to investigate other steps in themanufacturing process for the ability to remove TTV (e.g. byprecipitation, adsorption or chromatography or filtration steps) orinactivate TTV (e.g. by chaotropic salts or substances which intercalatein nucleic acids, or by irradiation with high-energy rays) using PCV. Itis additionally possible to establish the capacity for inactivatingand/or removing viruses also, for example, for additives in theproduction of pharmaceuticals, such as, for example, sera and otheringredients of media for cell cultures for producing recombinantproteins or monoclonal antibodies for affinity chromatography forpurifying and concentrating active ingredients.

The invention is explained in detail by the following examples:

EXAMPLE 1

The culture supernatant from a PK15 culture which had been maintainedfor many tissue passages and which showed, five days after passaging, apositive signal for PCV in the PCR was subcultured in the ratio 1:100 oncell cultures of various cell lines which had been freshly seeded out inT25 cell culture bottles.

The following permanent cultures of porcine cells were inoculated:

Fetal porcine kidney FPK Fetal porcine thyroid FPTh Fetal porcine testisFPTe Fetal porcine spleen FPSp Fetal porcine heart FPH Fetal porcineskin FPSk Porcine kidney PS

Ten days after inoculation, all the cultures apart from FPTh and FPSkshowed cytopathogenic changes (CPE). The cell culture supernatant washarvested from the CPE-positive cultures and stored at −80° C. untilused for further experiments.

A second passage of the CPE-causing agent was carried out on thehomologous cell cultures by inoculating these cell cultures with thecell culture supernatant from the first passage.

A distinct CPE was evident after only four days in the PS cells; withthe other cultures, the CPE was visible for the first time six daysafter inoculation. Using a specific PCR employing the abovementionedprimers it was possible to detect PCV in the PS cell culture showing aCPE, while no PCR signal was evident in the corresponding control cellswithout inoculation of the PK15 cell culture supernatant.

EXAMPLE 2

The PCV grown in the PS cell culture was quantified in the harvestedcell culture supernatant which had been centrifuged at low speed, bymeans of the end-point dilution method. The cell culture supernatant wasdiluted in 10-fold dilution steps and transferred to PS cell cultures inmicrotiter plates, and the PCV growth was evaluated as cytopathogeniceffect (CPE). Final reading of the test took place seven days after theinfection, and the virus titer (CCID₅₀—cell culture infective dose50%—in logio) was calculated by methods known to the skilled worker (6).Since circoviruses are non-enveloped viruses, as a check and to confirmthat the cytopathogenic effect is attributable to the growth of PCV inthe cell culture, part of the PS cell culture supernatant was treatedwith chloroform; this method is known to the skilled worker toinactivate enveloped viruses. Comparative titration of the untreated andchloroform-treated virus suspension in PS cells revealed no differencein titer (Table 1). This result, together with the specific PCR result,confirms growth of PCV in the cell culture, in particular in porcinecells.

TABLE 1 Quantitative determination of PCV grown in PS cells Virussuspension log₁₀CCID50/ml PS cell culture supernatant 7.8 PS cellculture supernatant, 7.6 chloroform-treated

EXAMPLE 3

Starting from the virus suspension of the PS cell culture—as describedin Example 2, freshly seeded cultures of various cells in T25 cellculture bottles were inoculated with a 1:100 dilution of the infectiouscell culture supernatant.

In total, three adaptation passages were carried out; i.e. thePCV-infected culture supernatant was put 1:100 v/v on freshly set upcultures of the homologous cell cultures ten days after infection. Thecultures with a cytopathogenic effect were tested in the PCR in order todemonstrate the identity of PCV. It surprisingly emerged from this thatPCV grows not only in the porcine cell cultures shown in Example 1 butalso in certain bovine and human cells, with development of acytopathogenic effect (Table 2).

TABLE 2 Demonstration of the growth of PCV in cultures of variousmammalian and human cells 1^(st) 2^(nd) 3^(rd) passage* passage*passage* CRFK = — — — Crandell Feline Kidney KFZI = — — — Feline KidneyMDBK = — — — Madin Darby Bovine Kidney BK C110 = — ? + Bovine Kidney,Clone 10 BK = — — — Bovine Kidney FRLu45 = — ? + Fetal Bovine Lung FROv= — — — Fetal Bovine Ovary FRMi = — — — Fetal Bovine Spleen FC-01 Ni = —— — Fetal Cynomolgus Kidney FRhK 4 = — — — Fetal Rhesus Kidney PH-2 = —— — Fetal Cynomolgus Kidney A549 = — — — Human Lung Ma23 = — — — HumanLung Mabt = ? + + Human Lung *Appearance of the CPE —no CPE ? CPEdoubtful/not pronounced + CPE pronounced

EXAMPLE 4

After in vitro growth of PCV in cell cultures had succeeded, thecapacity of sera from various mammals to neutralize PCV wasinvestigated. These investigations indicate in which mammals PCV grows,with subsequent seroconversion, or whether their sera contain antibodieswhich cross-react with PCV. However, it was not possible with thepresent experimental design to distinguish reliably between these twopossibilities. Various sera from individual animals (dog, cat, horse,pig, monkey) and pooled sera from several animals (cattle) orimmunoglobulin concentrates from human pooled plasma were tested in theneutralization test—antibody dilution and constant amount of virus(about 100 CCID₅₀). As Table 3 below shows, neutralizing antibodiesagainst porcine circovirus are detectable only in porcine sera and humanimmunoglobulin (e.g. Beriglobin 7).

TABLE 3 Detection of PCV-neutralizing antibodies in sera from variousspecies Number of sera Number of PCV- Species investigated positive seraBeriglobin* batches 24 24 Dog 10 0 Cat 10 0 Horse 10 0 Bovine 10 0 Pig10 10 Monkey 10 0 *purified human gamma-globulin concentrate fromdonated pool NT index > 0.5 was assessed as positive

EXAMPLE 5

The PCV-neutralizing and non-neutralizing antibodies can also bedetected with other in vitro methods known to the skilled worker, e.g.with enzyme immunoassays (EIA) . Adsorption of PCV onto a solid phase(e.g. polystyrene, nylon or cellulose) with subsequent incubation of thesera to be investigated and further incubation with enzyme-labeled,secondary antibodies directed against the primary antibodies present inthe sera leads to quantification of antibodies directed against PCV inthe serum to be investigated.

PCV-containing cell culture supernatant from PS cells was prediluted1:100 in 0.1 M NaOH and pipetted in a geometric dilution series into anELISA microtiter plate (dilution buffer 0.05 M Na₂CO₃, pH 9.6). Thecolor intensity was measured by methods known to the skilled worker forblocking the plate, and incubating with serum to be investigated andlabeled antibodies directed against the serum to be investigated (Table4). The example shows that antibodies against porcine circovirus aredetectable only in porcine sera and human immunoglobulin concentrates.

TABLE 4 ELISA of various sera for detecting antibodies against porcinecircoviruses (extinction) Human immunoglobulin Virus concentrate Porcinedilution (Beriglobin P) serum Equine serum 1:200  >2.000 >2.000 0.3861:400  1.983 >2.000 0.426 1:800  1.168 1.852 0.189 1:1600 0.726 1.2330.253 1:3200 0.268 0.706 0.335 1:6400 0.381 0.457 0.129

EXAMPLE 6

In the manufacture of therapeutic compositions or substances intended tobe employed for their manufacture from biological material it isnecessary to inactivate or remove viruses which are potentially present.For this reason, the thermal stability of porcine circovirus was testedin various media in a further series of tests at 60° C. (pasteurizationin aqueous solution):

a) in Eagle's minimal essential medium (EME medium)

b) in 5% strength human serum albumin solution (HSA)

c) in pasteurization buffer for blood coagulation (FVIII) products(aqueous solution stabilized with sucrose and glycine).

For this purpose, PCV was produced as in Example 2, added (spiked) 1:11v/v to the three media mentioned above and heated in a water bath at 60°C. After the stated times, samples were taken for titration of theremaining virus and titrated on PS cells.

Final reading of the titer took place 7 days after setting up the testby observing the cytopathogenic effect under the microscope; the resultsare shown in Table 5.

As the results show, PCV is unstable to physico-chemical parameters suchas elevated temperature. Stabilizers added to the pasteurization bufferfor factor VIII products in order to stabilize this factor during thepasteurization (heat treatment at 600° C. in stabilized aqueoussolution) likewise stabilize PCV to a certain extent. It is thuspossible to use circoviruses for investigating the capacity of a methodof manufacture or of a diagnostic aid to inactivate and/or removecircoviruses or related viruses.

TABLE 5 Virus titer (log₁₀ CCID₅₀/ml) after incubation of PCV in variousmedia at 60° C. PCV in 5% PCV in factor PCV in cell human VIIIPasteurization culture serum pasteurization time [h] medium albuminbuffer 0 5.9 5.8 5.8 1 ≦1.5 1.8 4.9 2 ≦1.5 ≦1.5 3.5 4 ≦1.5 ≦1.5 2.9 6≦1.5 ≦1.5 1.8 8 ≦1.5 ≦1.5 ≦1.5

COMPILATION OF LITERATURE

1. Handa, A. et al., Prevalence of the newly described human circovirus,TTV, in United States blood donors.—Transfusion 40, 245-251 (2000)

2. Tischer, I., Bode, L., Apodaca, J., Timm, H., Peters, D., Rasch, R.,Pociuli, S. and Gerike, E. “Presence of antibodies reacting with PorcineCircovirus in sera of humans, mice and cattle”; Arch. Virol. 140,1427-1439 (1995)

3. Morovsov,, I., Sirinarumitr, T., Sorden, S. D., Halbur, P. G.,Morgan, M. K., Yoon, K.-I. and Paul, P. S. “Detection of a novel strainof Porcine Circovirus in pigs with postweaning multisystemic wastingsyndrome” J. Clin. Microbiol. 36, 2535-2541 (1998)

4. Hinrichs, U. et al., Erster Nachweis einer Infektion mit dem porzinenCircovirus Typ 2 in Deutschland.—Tierärztliche Umschau 54, 255-258(1999)

5. Allan, J. S. “Nonhuman primates as organ donors?” Bull. WHO 77 (1),62-63 (1999)

6. Kärber, C. “Beitrag zur kollektiven Behandlung pharmakologischerReihenversuche” Arch. Exp. Path. Pharmak. 162:480-487 (1931)

7. Mankertz, A. et al., 1997. Mapping and characterization of the originof DNA replication of porcine circovirus. J. Gen. Virol. 71:2562-2566.

4 1 21 DNA Artificial Sequence Description of Artificial Sequence Primer1 gagaggaagg tttggaagag g 21 2 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 2 ccactggctc ttcccacaac c 21 3 22 DNAArtificial Sequence Description of Artificial Sequence Primer 3ggtgaagtgg tattttggtg cc 22 4 24 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 ctatgacgtg tacagctgtc ttcc 24

What is claimed is:
 1. A method for growing circoviruses, whichcomprises the steps of a) obtaining circoviruses from an infected firstcell culture, b) inoculating a second cell culture with thecircoviruses, c) making one or more passages of the second cell culture,d) analyzing said second cell culture for a cytopathogenic effect (CPE),and e) harvesting the circoviruses from the CPE positive cultures. 2.The method as claimed in claim 1, wherein the circoviruses are porcinecircoviruses.
 3. The method as claimed in claim 1, wherein the secondcell culture comprises cells of porcine, bovine or human origin.